The enzyme citrate lyase (EC4.1.3.6) is regarded as a key enzyme of anaerobic citrate degradation and can accordingly be isolated from a number of different prokaryotic cells. The enzyme catalyses the cleavage of citrate into acetate and oxaloacetate. Furthermore it is known that the enzyme complex of the citrate lyase enzyme that has been best examined to date from Klebsiella pneumoniae (formally: Klebsiella aerogenes) is composed of six copies of each of three different subunits and namely an xcex1, xcex2 and xcex3 subunit, of a molecular weight of about 550,000 Dalton. In addition it is known that the catalytically active center is located in the xcex1 and xcex2 subunit, whereas the xcex3 subunit has the binding site for the prosthetic group 2xe2x80x2-(5xe2x80x3phosphoribosyl)-3xe2x80x2-dephospho CoA. This prosthetic group is bound to the serine residue 14 via a phosphodiester bond.
The citrate lyase enzyme is required in high purity for most applications which are primarily for clinical chemistry and food analysis. Hence the aim is to over-produce the enzyme in an active form in certain host cells by recombinant methods and to isolate it from these cells. Such a process has not yet been described or made known in other ways. Hence citrate lyase is nowadays usually isolated from Klebsiella pneumoniae cells which had been cultured under anaerobic conditions using citrate as the only carbon and energy source. The citrate lyase genes from Klebsiella pneumoniae have been cloned and sequenced (M. Bott and P. Dimroth, Mol. Microbiol. Vol. 14, 347-356 (1994)). These genes are part of the citC operon which is composed of the five genes citCDEFG. The citC gene codes for citrate lyase ligase which catalyses the formation of an acetyl thioester. The genes citD, citE and citF code for the gamma, beta and alpha subunit of citrate lyase. The protein coded by citG is involved in the biosynthesis of the prosthetic group. Furthermore it is known that the citC operon is induced in the absence of oxygen and in the presence of citrate and Na+ ions; moreover the expression is strongly dependent on the citA/citB regulation system (M. Bott et al., Mol. Microbiol. Vol. 18, 533-546 (1995); M. Meyer et al., J. Mol. Biol. Vol. 269, 719-731 (1997)).
Expression of the genes coding for citrate lyase from Klebsiella pneumoniae which would preferably be carried out in prokaryotic cells such as E. coli for practical reasons, results in an inactive but nevertheless soluble form of the enzyme (M. Bott and P. Dimroth, Mol. Microbiol. Vol. 14, 347-356 (1994)). The recombinant apo-citrate lyase enzyme can be activated to form the holo-enzyme by subsequent addition of acetyl coenzyme A which is known as a substituent for the acetyl thioester of the native prosthetic group 2xe2x80x2-(5xe2x80x3-phosphoribosyl)-3xe2x80x2-dephospho CoA. However, such an additional activation measure is complicated and laborious. Moreover the necessity to add acetyl CoA is unsuitable for the commercial distribution of citrate lyase or the apo form since the substance decomposes when stored for long periods at 4xc2x0 C.
Hence the object of the invention is to provide a recombinant, soluble and at the same time active holo-citrate lyase which eliminates the disadvantages of the known methods.
The object is achieved by a process for the production of a protein with citrate lyase activity by expressing a suitable plasmid in a host organisms whereby the plasmid contains the information of a gene cluster composed of at least six genes and an inducible promoter. The genes comprising the gene cluster code for certain subunits of the protein with citrate lyase activity and/or for a component which participates in the biosynthesis of the complete enzyme. In particular a suitable plasmid contains the genes citC, citD, citE, citF, citG and a DNA fragment that can for example be obtained from E. coli which is located between the genes citF and citG on the E. coli citrate lyase gene cluster. The genes citD, citE and citF code for the corresponding xcex3, xcex2 and xcex1 subunits of the enzyme and have molecular weights of about 11,000 Dalton, 32,000 Dalton and 55,000 Dalton. According to the invention it is preferred that one of the genes represents a DNA fragment which codes for a protein containing the motif G(A)-R-L-X-D-L(I)-D-V. A corresponding DNA fragment is particularly preferred which codes for a protein with a molecular weight of about 20,000 Dalton.
In addition it has proven to be advantageous when one gene and optionally a further gene fused to the first gene of the genes comprising the gene cluster is derived from a different organism than the other genes. In particular it has proven to be advantageous when the DNA fragment citX or genes homologous to citX located between citF and citG on the E. coli citrate lyase gene cluster are derived from E. coli, Klebsiella pneumoniae, Haemophilus influenzae or Leuconostoc mesenteroides and when one or several of the other genes are derived from the microorganism that is specific for the isolated protein having citrate lyase activity which is for example Klebsiella pneumoniae. In Haemophilus influenza, Leuconostoc mesenteroides (S. Bekal et al., J. Bacteriol. Vol. 180, 647-654 (1998)) and Leuconostoc paramesenteroides (M. Martin et al., FEMS Microbiol. Lett. Vol. 174, 231-238 (1999)) the genes citX and citG occur in a fused form. Thus corresponding fusion genes contain the information of two genes. The resulting proteins have a molecular weight of about 52,000 Dalton, have the activities of E. coli CitX and CitG and are thus bifunctional. In the absence of the citX gene or of a gene homologous to citG or of a corresponding citX fusion gene, only the low-molecular apo form (MW 12,000 Dalton, SDS-PAGE) but not the holo form of citrate lyase (MW 14,500 Dalton, SDS-PAGE) could be detected after expression.
According to the invention prokaryotes as well as eukaryotes have proven to be suitable as the host organism. The fact that a soluble active citrate lyase can now be produced in prokaryotes such as e.g. E. coli in a simple manner and in adequate yields without additional activation measures is a major advantage.
Hence it was possible to show that by cloning the entire E. coli citCDEFXG gene cluster under the control of an inducible promoter such as e.g. the lac, lac UV5, T5, tac or T7 promoter, an active enzyme can be expressed having citrate lyase activity even under non-oxygen limiting conditions. Cell extracts containing appropriate expression plasmids result in citrate lyase activities of about 4 to 5 U/mg protein in the cell-free extract whereas cells without recombinant citrate lyase have no citrate lyase activity when grown aerobically.
In addition the invention concerns the simultaneous expression of the citCDEFG gene cluster from Klebsiella pneumoniae and of the citX gene obtainable from E. coli by which means it is possible to obtain a corresponding citrate lyase in an active form even in prokaryotes and in particular in E. coli. 
By this means it was possible to achieve an activity of about 8 U/mg total protein in a cell-free extract under aerobic growth conditions.
The holo-enzyme is purified by methods known to a person skilled in the art. About 100 to 120 xcexcg soluble protein with citrate lyase activity can be obtained from about 1 g of cells (wet weight) using the process according to the invention. The protein determination was carried out according to P. K. Smith et al., Anal. Biochem. Vol. 150, 76-85 (1985) using ovalbumin as a standard. The specific activity of the citrate lyase is ca. 70 U/ml protein (M. Single and P. A. Srere, J. Biol. Chem. Vol. 251 (10), 2911-2615 (1976). The activity of the holo-enzyme that can be obtained by the process according to the invention is thus ca. 0.5 to 3-fold higher than the activity that was achieved with acetyl CoA and apo-citrate lyase.
Hence the process according to the invention provides for the first time a recombinant protein with improved citrate lyase activity that is both soluble and active.
Furthermore the invention concerns a test kit for the determination of citric acid which is composed essentially of the following components: a protein obtainable by the process according to the invention with citrate lyase activity, at least one protein with hydrogen-transferring activity, nicotinamide-adenine dinucleotide or an appropriate derivative in a reduced form and optionally suitable stabilizers, activators and/or substances to avoid or reduce interferences i.e. components or reactions which mask or interfere with the actual reaction as well as suitable buffer solutions. In particular L-malate dehydrogenase and L-lactate dehydrogenase come into consideration as proteins with hydrogen-transferring activity. Those substances, additives or measures which help to avoid or at least to delay the degradation of a property or activity that is important for the determination are in principle suitable as stabilizers. Especially when only small amounts of sample material are available or if the samples are very dilute it can be advantageous to add activators.
An additional subject matter of the invention is the use of the recombinant soluble protein with citrate lyase activity to determine citric acid in clinical chemistry, food analysis and as a purity test for cosmetics. In clinical chemistry a corresponding enzymatic test is used primarily to examine fertility and for therapeutic monitoring of patients with kidney stones. In food analysis the most important application is analysis of wines and fruit juices.
The enzymatic method is based on the cleavage of citrate by the enzyme citrate lyase in the presence of Mg2+ ions to form oxaloacetate and acetate. In the presence of hydrogen-transferring enzymes such as L-malate dehydrogenase and L-lactate dehydrogenase, oxaloacetate and its decarboxylation product pyruvate are reduced by reduced NADH or NADPH to form L-malate and L-lactate. The amount of NADH or NADPH is proportional to the amount of citrate and is measured at 334 nm, 340 nm or 365 nm.
Hence the invention also concerns a corresponding test kit for the determination of citric acid which, apart from suitable buffer solutions, contains a recombinant protein with citrate lyase activity, one or several hydrogen-transferring enzymes and a nicotinamide adenine dinucleotide or a corresponding derivative in a reduced form and optionally suitable stabilizers such as thiol reagents.